1. Field of the Invention
The invention relates to a signal peptide for the production of recombinant proteins.
2. Background Art
The large-scale industrial production of recombinant proteins is of increasing importance to the biotechnology and pharmaceutical industry. In general, recombinant proteins are produced either in mammalian cell culture or in microbial systems. Compared to mammalian cell culture, microbial systems have the advantage that in this manner recombinant proteins can be produced in a shorter time and with lower costs. Hence bacteria, preferably those of the genus Escherichia, more preferably E. coli, are most suitable for the production of recombinant proteins. In E. coli, recombinant proteins can in principle be produced in various ways:    1. Intracellular production as soluble protein;    2. Intracellular production as inclusion bodies;    3. Secretion into the periplasm or into the nutrient medium.
The production process for the recombinant protein always consists of two parts. The first part is the fermentation, which leads to the crude product. In this case, the fermentation result, which contains the recombinant protein and also contaminating host proteins, is described as the crude product. The second part of the production process comprises the purification of the recombinant protein starting from the crude product.
In addition to the production costs of the crude product which is present directly after the fermentation as a mixture containing the recombinant protein and host proteins, the labor and costs for the production of the recombinant protein are also to a considerable extent determined by the costs of purification of the crude product to the desired recombinant protein. The purification is in most cases performed over several stages by means of chromatographic procedures. Purification from contaminating host proteins, some of which are immunogenic or toxic, is an important aspect.
The secretion of proteins in E. coli in most cases takes place via the so-called sec pathway (Driessen et al., 1998). This system is responsible for the export of certain bacterial proteins. The genes for these proteins each have a so-called signal sequence at the 5′ end. During protein synthesis, this is translated into a signal peptide and effects the secretion of the protein through the cytoplasmic membrane. After secretion, the signal peptide is removed by the enzyme signal peptidase and the mature protein is released.
The sec system can also be used for the secretion of recombinant, for example heterologous, proteins (Lee et al., Methods in Molecular Biology 308, 2005). For this, the recombinant gene for the recombinant protein to be produced is linked with a signal sequence (“in-frame fusion”), which results in the production of a signal peptide-protein fusion product. The signal peptide encoded by the signal sequence mediates the secretion of the recombinant protein across the cytoplasmic membrane into the periplasm by means of the bacterial sec system. In this, the signal peptide is cleaved off at the cleavage site between signal peptide and the recombinant protein, and the desired recombinant protein is obtained in the periplasm. The recombinant protein can then be purified from the periplasm.
Compared to the other production processes, secretion offers the advantage that the recombinant protein is obtained directly as native, soluble, correctly folded protein, which in contrast to the “inclusion body” process does not have to be denatured and again renatured, a step which is attended by major losses in yield. Moreover, in this case the crude product is contaminated with fewer host proteins compared to intracellular soluble production, since the periplasm of bacteria contains far fewer host proteins than the cytoplasm.
Under certain conditions or in certain bacterial strains, the recombinant protein is released from the periplasm into the nutrient medium (e.g. Ray et al., 2002; EP0338410B1; Nagahari et al., 1985; Yang et al., 1998; EP0677109B1) and can be purified from this.
Compared to secretion into the periplasm, secretion of the proteins into the nutrient medium offers an advantage that the protein is then present in still purer form. Moreover as the first purification step, laborious preparation of the periplasm or disintegration of the cells is unnecessary, but rather the much simpler and more reproducible removal of the whole cells.
As aforesaid, for the secretion of a protein to be produced, the gene coding for it is linked with a signal sequence, which has the effect that the protein to be produced is initially produced as a fusion product with the signal peptide encoded by the signal sequence. This signal peptide effects the secretion of the protein produced.
Signal peptides are made up of three regions: the N-terminal N region (1-5 amino acids) as a rule contains one or more amino acids, which bear a positive charge. The H region lying in the middle mostly consists of 7-15 amino acids, many of which are hydrophobic. The C region as a rule comprising 3-7 amino acids mostly contains neutral, short-chain amino acids (A, G, S, T or C) at position −1 and −3 before the cleavage site.
Various signal sequences and the corresponding signal peptides are described in the state of the art, e.g. phoA, ompA, pelB, ompF, ompT, lamB, malE, staphylococcal protein A and stII (Choi & Lee, 2004; EP0396612B1). The signal peptide of the cyclodextrin glycosyltransferase (CGTase) from various strains, such as for example Klebsiella oxytoca (Klebsiella pneumoniae M5a1), and the use thereof for the secretion of CGTase in E. coli strains is described in U.S. Pat. No. 5,395,927. Also described (EP0448093B1) is the fact that a recombinant protein, such as for example a hirudin derivative, can be produced and secreted in E. coli strains through fusion of the gene for the recombinant protein with the signal sequence of the CGTase. In the case of a specific hirudin derivative, this leads to a yield of 250 mg/l in a shaker flask culture and 2.63 g/l in a fermentation. EP0448093B1 however also describes the fact that with another recombinant protein yields of only up to 25 mg/l were obtained. The signal peptide of CGTase is like all other known signal peptides—not capable of mediating secretion of any recombinant protein in equally high yields. Since every recombinant protein is encoded by its own DNA sequence and in particular the DNA sequence at the transition point between signal sequence and the sequence coding for the recombinant protein is therefore different, as a rule an optimal signal peptide must be found for each recombinant protein.